Inflammatory cells dynamics control neovascularization and tissue healing after localized radiation induced injury in mice

Local overexposure to ionizing radiation leads to chronic inflammation, vascular damage and cachexia. Here we investigate the kinetics of inflammatory cells from day (D)1 to D180 after mouse hindlimb irradiation and analyze the role of monocyte (Mo) subsets in tissue revascularization. At D1, we find that Mo and T cells are mobilized from spleen and bone marrow to the blood. New vessel formation during early phase, as demonstrated by ~1.4- and 2-fold increased angiographic score and capillary density, respectively, correlates with an increase of circulating T cells, and Mohi and type 1-like macrophages in irradiated muscle. At D90 vascular rarefaction and cachexia are observed, associated with decreased numbers of circulating Molo and Type 2-like macrophages in irradiated tissue. Moreover, CCR2- and CX3CR1-deficency negatively influences neovascularization. However adoptive transfer of Mohi enhances vessel growth. Our data demonstrate the radiation-induced dynamic inflammatory waves and the major role of inflammatory cells in neovascularization.

VEGF mRNA dynamics (Fig. 5B) do not match protein dynamics (Fig. 5D). Furthermore, the Vegf mRNA peaks at D90 (Fig. 5B), but the capillary density is significantly suppressed (Fig. 5F), a contradiction. The authors must explain these contradictory data. Also, the Vegf protein cannot be considered biologically meaningful if there is no significant increase or decrease (Fig. 5D).
In D3, the number of Mohi and Molo in the blood of ccr2-/-mice, but not cx3cr1-/-mice, was significantly lower than in wild-type mice (Fig. 6A), what was found and considered? The authors should also indicate whether there were changes in leukocyte infiltration into the muscle with ccr2-/-and cx3cr1-/-mice.
What about BM-derived EPC infiltration, which is important for angiogenesis (DOI: 10.1172/JCI43027), into the injured muscle throughout the experimental period?
The scRNAseq experiment will address some of the questions that reviewers and readers may have.
Regarding the third paragraph on page 9, I wonder if a Bayesian approach is appropriate for this study. Why not actually conduct a mouse experiment and use various M1 and M2 markers to prove it? Furthermore, the fourth paragraph is too descriptive.
"In addition, Molo numbers in cx3cr1-/-mice were …" in page 10, line 18-19. Since no significant decrease was observed, this sentence should be omitted to avoid misleading the reader.
Reviewer #3 (Remarks to the Author): The authors investigated the role of inflammatory cell populations in response to radiation-induced injury of the muscle at early and late phases post-irradiation. They identify the dynamic nature of the immune response and recruitment of key responders to the site of injury and the role they play in both early neovascularization and injury progression establishing a relationship between specific immune cell populations and vascularization states. The presented work is very well done and is of great interest and relevance to the radiation therapy field.
There are some technical points, however, that should be addressed as well as some questions regarding some of the results: 1. There are discrepancies between figures referred to in the text and the actual figures themselves. In particular, referring to statistical significance in the text but lacking significance markings in the graphs. Also, they sometimes refer to incorrect figure labels within the text. a. Figure 2D is listed as 1D in text-in the text it indicates Mo hi, Mo lo and Neutrophils peaked at D1, D5 and D60 however in Figure 2 there is only statistical significance at D1 b. Figure 4C (T lymphocytes)-in the text they state The number of CD3 positive lymphocytes increased by 39-fold at D7, 24-fold at D14 and 18-fold at D21 however in the graph there is no D21, only D30 c. Figure 6E is referred to as 6D in text 2. There are acronyms such as NIR that have not been defined before their use and the manuscript 3. Material and Methods-Irradiation: The author should specify the size of the irradiation field and/or the portion of mice body surface irradiated. 4. Material and Methods-Mice are all male. The author should justify their specific choice and discuss whether they are expecting different results in the case of employing female mice. 5. Material and Methods-Flow cytometry mix of Abs: The author are talking about one mix but they should divide the Abs in groups according to the different combinations used in the different panels employed. Regarding results/discussion: 6. NIR controls are referred as littermates of the irradiated mice. However, there are studies showing the existence of bystander effects from irradiated animals to non-irradiated animals when they are cage mates, especially after high dose RT. The author should address this point in the discussion. 7. How do the authors explain such an early influx of M-2 like MFs in the muscle? In the discussion they mention the importance of this pro-angiogenic MFs subpopulation in the resolution of Myocardial infarction (MI), however in MI but also in other types of sterile injuries the content of M2-like MFs peaks at 5/7 days post-injury not earlier. Also, because they are gradually differentiating from M1-like Mo/MFs which are the first ones reaching the injured tissue. Author should discuss their findings in relation to this and hypothesize about the reason of the difference between them and what is known in the literature. 8. Furthermore, I would like them to comment on the sustained levels of neutrophils, Mo(hi) and Mo(lo) in the blood (depicted in Figure 2C) and provide a hypothesis or details on why the fluctuations in these cells observed in the spleen and muscle are not reflected in the blood. This report has analyzed the dynamics of inflammatory cells and neovascularization in radiationinduced muscle injury in mice. However, it is overall too descriptive and is recommended for rejection.

1/ This manuscript needs to be edited in English.
The manuscript has been edited by a native English speaker.

2/ Overall, the use of paragraphs should be modified according to the journal's requirements.
As requested, the paragraphs have been modified according to the journal's requirements 3/ The importance of the radiation-induced muscle injury model should be described in more detail in the first paragraph of Introduction.
Local overexposure to ionizing radiation has severe health consequences, especially when the absorbed dose exceeds 25 Gy and leads to tissue necrosis 1,2 .
Clinical signs of a patient exposed to ionizing radiation have been already described and it has been mentioned extensive necrosis in the right limb, intense pain, multiple infections and ankyloses of the right hip and knee 1 . Clinically, the symptoms appear days after the exposure and the lesion is described as dynamic, with a progressive extension of the radio-induced damage associated with ischemic, inflammatory and necrotic processes [3][4][5] . At the molecular level, nuclear DNA damage, oxidative stress caused by reactive oxygen and nitrogen species and mitochondrial dysfunction with perturbations in oxidative metabolism have been previously described 6,7 . In the particular case of radiation muscular injury, the evolution of the damage in patients has been associated to a decrease of muscular function 8 .
As requested, we have clarified the importance of the radiation-induced muscle injury model and with the changes highlighted in yellow in the manuscript: page 3, line [16][17][18][19][20][21] In experimental model, it has been shown that the numbers of both myonuclei and satellite cells per myofiber were decreased in a dose-dependent manner 9 . Moreover, a single irradiation dose of 18 Gy block muscle regeneration by promoting lethality of myoblasts 10 . Studies of muscular pathology using 2 more than 25 Gy irradiation dose, have shown morphological alterations, hemorrhage, necrosis, inflammation, fibrosis and mitochondria destruction [11][12][13][14] . In our preclinical model of 120 Gy local radiation hindlimb in nude mice (unpublished data) we demonstrated a characteristic muscular pathological feature including severe atrophy, central nuclei, fibrosis and inflammatory infiltrates.
Furthermore, an acute damage to the microvascular network of the irradiated muscle was evident with a decrease in the endothelial cells number and an abnormal cellular morphology. These findings were in accordance to the results obtained in a victim from accidental exposure to high dose ionizing radiation. Additionally, the irradiated mouse muscle presented a fiber-typing switch with an increase in the type IIA and IIX fast fibers compared to controls. Some embryonic MHC positive fibers. After, high dose exposure to radiation, the tissue maintains a deleterious T cells pro-inflammatory response during the long term follow up until 6 months.

4/ Lane 7 in page 4 mentions the ischemia model but should describe in detail what it has in common
with the radiation model.
As requested, we describe in detail the similarities between ischemia disease and radio-induced local injury (RLI) and the changes highlighted in yellow in the manuscript: page 3-4, line 17-2. Ischemia is the common process in both diseases RLI and cardiovascular disease. In ischemic disease, insufficient organ perfusion following thrombotic vessel obstruction of the feeding artery is a major determinant of post-ischemic remodeling 15 . However, exposure of mammalian cells such as endothelial cells to ionizing radiation leads primarily to DNA damage-induced cell death 16 . Ischemia is characterized by vascular damage/rarefaction and inflammation resulting in fibrosis characterized by collagen-based scar 17 . The ischemic tissue response is based on four principal processes, vasculogenesis, angiogenesis, arteriogenesis, and collateral growth, which contribute to tissue repair and remodeling during acute and chronic ischemic vascular diseases 18 . These processes result from hemodynamical forces changes within the vascular wall leading to modification of the vascular homeostasis 19 18 .
As requested, we added this sentence page 5, line 7-8: Therapeutic modulation of the inflammatory response may hold promise to improve reparative response for the prevention of postischemic disease.

5/ The authors should discuss how the results of this study will ultimately be applied to humans.
The aim of this study is to understand the chronologic events in early and late time post-IR and we focus on the role of Mo/ Mɸ. We showed that ionizing radiation induces an early phase characterized by a pro-inflammatory recruitment and infiltration of CD4-, CD8-T cells, Mo hi and M1-like leading to the neovascularization process to prevent tissue hypoxia. Afterwards, we observed a late phase supported by a positive correlation between the decreased recruitment of anti-inflammatory Mo lo and differentiation into M2-like, vascular rarefaction and cachexia characterized by sustained proinflammatory response. In this context, we would like to propose a personalized medicine and possibility to use Mo/Mɸ as biomarker to i) predict tissue complication after radiation exposure and/or to ii) determine the therapeutic strategy.
In the discussion of the manuscript, we suggest that Mo/Mɸ could be used as biomarkers to predict the radiation induced tissue complication. In fact, Mo/Mɸ have been already published as a good biomarker in a clinical study, conducted on a cohort of 49 patients included in breast cancer radiotherapy protocol, which showed that the increase of mRNA RG1 level expression in Mɸ 24 hours after a first session of radiotherapy, was correlated with the risk of developing severe erythema 8 weeks after the end of radiotherapy 20 . Finally, in patients with radiation-induced mucositis, the number of Mɸ has been associated with the severity of the disease 21 .
Moreover, we propose to optimize the medical management by a personalized regenerative treatment based on the modulation or reprogramming of the anti-inflammatory/pro-inflammatory phenotype of Mo/Mɸ. Several studies have shown that nanoparticles injection loaded immunomodulatory drugs lead to reprogramming the anti-inflammatory/pro-inflammatory function of Mo/Mɸ to repair tissue or for cancer treatment [22][23][24][25] . For example, Jiang et al. 26 , in CCl4-induced mouse liver injury model, showed that nanoparticles loaded with tripolyphosphate dynamically regulated M1 into M2 macrophage reprogramming and relieved the liver injury. Finally, in a colon cancer mouse model, M2 macrophages treated with PEGylated liposomes containing IFN-γ expressed elevated NO and decreased arginase levels, promoted M2 to M1 polarization and significant antitumor responses 27 . In our condition, we would suggest nanoparticles injection, that will potentially carry antiinflammatory or pro-inflammatory function (depending on the inflammatory state of the patient after blood collection or tissue biopsy) with the aim to modulate the inflammatory reaction and stimulate the regenerative tissue process.

6/ In the first paragraph of page 6, the authors should describe the details of the scoring criteria.
As requested, we give more details for the scoring and we added the changes highlighted in yellow in the manuscript in materiel and methods page 17, line 17-20. A score between 0 (normal) and 1 (max injury) was assigned to each parameter regarding wound extent, ulceration, moist desquamation and limb retraction (see Figure below), resulting in a total injury scoring during the wound development and healing process each week post-irradiation.

7/ In line 15 of page 6, "leucocytes"  "leukocytes".
We modified the word. As requested, we changed the meaning of the sentence by: we did not observe monocyte accumulation in muscle, suggesting they differentiated in macrophages in line 17-18 of page 6 and highlighted in yellow in the manuscript 9/ What is "NIR"? Abbreviations must be explained.
We added the complete name of Non-irradiated (NIR) in the manuscript page 6 line 18 10/ References are required after the sentence on page 7, line 21.
As requested, line 21, page 7 a reference has been added and highlighted in yellow in the manuscript. As requested, to better understand the kinetics of inflammatory response and the healing process of the radiation-induced muscle injury, we performed specific staining with visualization of macrophages and T cells after using a CD68 or CD3 antibody respectively. CD68-and CD3-positive cells were counted in randomly chosen fields with the use of Image J software (NIH). Additionally, the sections were stained with hematoxylin and eosin (H&E) to analyze muscle structure. During many months to years, clinical signs are characterized by intense pain, multiple infections and ankyloses of the right hip and knee 1 , successive and unpredictable inflammatory waves over the first few days to years after irradiation, and these lead to horizontal and vertical extension of tissue injury including vascular rarefaction and muscle cachexia 2 .
6 Experimental data: with the clinical hindlimb radio-induced injury, the mouse model was used in order to mimic the clinical features with inflammatory waves, vascular rarefaction, cachexia injury and healing aspects up to 6 months post-irradiation as observed in the patient. We showed previously on unpublished results from the lab, on Nude immunodeficient mice, an increase in the injury score two weeks post-irradiation (40 or 60 Gy), with a return to a non-irradiated situation maintained for up to 6 months after irradiation, compared to BalB/c (immunocompetent).
Moreover, in immunocompetent mice a second wave of injury score was observed at In our study, we define the acute and late phases in function of the vascular process. Therefore, we determined D0-30 as the early phase corresponding to the neovascularization process and proinflammatory cells recruitment, and D60-180 as the late phase characterized by vascular rarefaction correlated to recurrent and chronic pro-inflammatory phenotype. Irradiation disease are really uncommon regarding inflammatory waves compared to other diseases such as myocardial infarct or peripheral occlusion disease. In cardiovascular ischemic disease, during the first acute phase (hours-4 days), it has been shown a pro-inflammatory recruitment, vascular regeneration and, subsequently a late phase (5-7 days after the ischemic event) corresponding to a switch and anti-inflammatory 7 recruitment and tissue regeneration 28 29 30 . In these diseases the acute and late phase are determined in function of the inflammatory swicht pro-vs anti-inflammatory process. Moreover, the main cells infiltrated in the tissue, after MI, corresponded to neutrophiles, Mo at D3 post-MI compared to T cells infiltration and returned to basal level at D7 or D14 post-MI 17 . After ionizing radiation, we do not observe the remodeling phase caracterized by the switch from pro-inflammatory to anti-inflammatory cell phenotype , but the maintenance of the pro-inflammatory response during a long time period after exposure. That is the reason why we decided to focus on the vascular process to establish the early and late phases. Moreover, in irradiated muscle the main cells infiltrated is corresponding to T cells and stay up until the end of experiment. This study highlights the pro-inflammatory waves after exposure and their effect on the vascular process at long term. That is the reason why we decided to define the acute and late phases, by taking into consideration the timeline of this study and the associated pathophysiological process, too.

D/ In addition, the meaning of the bimodal infiltration of DCs and lymphocytes into muscle (graphical abstract) should be examined and clarified.
The bimodal infiltration of DCs and lymphocytes into muscle is a consequence of radiation exposure.
In the study described in reference 1, such successive inflammatory waves were observed on the patient over the first few days, weeks to years after irradiation. In this study, the preclinical model developed tries to mimic the clinical situation (characterized by chronic inflammatory waves, vascular 8 rarefaction, and cachexia). Based on the graphical abstract, we can observe that the level of LT and DC peaks at different time points and never return to a non-irradiated condition, which explains the state of a proinflammatory tissue and vascular damage with no regeneration process. This is the specificity of the tissue response in irradiation condition.
The difference with cardiovascular ischemic diseases (hindlimb or MI), is that the inflammatory cells peaked at D3 and then return to basal level from 7 to 14 17 28 .

15/ Overall, there appears to be inconsistency and discrepancy in the various longitudinal data. For
example, cx3cl1 mRNA peaks at D90, but lymphocyte infiltration peaks at D7 and is baseline at D90.
Furthermore, the peak of eNOS protein expression is D30, while the peak of angiogenesis is D14.

A/ Overall, there appears to be inconsistency and discrepancy in the various longitudinal data. For example, cx3cl1 mRNA peaks at D90, but lymphocyte infiltration peaks at D7 and is baseline at D90.
According to the kinetic of evolution of CX3CL1, we observed -The tendency to increase of mRNA expression level of CX3CL1 associated with a significant increase of CD3 positive cells in the muscle from D7 to D30 post-IR suggesting the recruitment of T cells from the blood into the muscle.
-The increase of T cells in the blood from D7 to D30 (cytometry estimation Figure 3B) associated with an increase of T cells in the muscle (cytometry estimation Figure 3C) and confirmed by T cells staining in the muscle (figure 4C) suggesting the recruitment of T cells from the blood into the muscle.
Concerning the peaks observed at D90 with no effect on the T Cells infiltration in the muscle. We suggest several hypotheses: -during the late phase, we observed a production of T cells in the spleen ( Figure 3A) and suggest a blocking of their egress from the spleen to the blood (as no significant increase of these in the blood). (Figure 3A, 3B) -the lack of presence of T cells in the blood could explain the absence of the recruitment in the muscle whatever the peak of CX3CL1 at D90 post-IR.

B/ Furthermore, the peak of eNOS protein expression is D30, while the peak of angiogenesis is D14.
The angiogenic process results of multiple pro-angiogenic factors and in this study, we analyzed eNOS and VEGF mRNA and protein known to be some of the key players in neovascularization process to repair ischemic tissue. Regarding the kinetic of evolution of these two actors, our results do not allow us to explain the stimulation of the vascularization process at the different time points corresponding to the protein analyses. We agree that a more accurate evaluation of the eNOS protein (additive time 9 points) could be more informative and support our hypothesis on the involvement of eNOS in the angiogenic process as described in several papers. eNOS has been already published to play a major role in healing process 31 . Additionally we could suggest the involvement of other factors such as, VEGF-B 32 , b-FGF 33,34 or Ang 35 largely described in the literature, for their major role in the stimulation of angiogenesis (we did not investigate in this study).

16/ Since the authors focus on angiogenesis, intramuscular angiogenesis should be shown
histologically (e.g., immunostaining with anti-cd31) in addition to microangiography to facilitate the reader's understanding.
We totally agree with the reviewer on the importance to evaluate intramuscular angiogenesis.
Different studies assessed angiogenesis using lectin as an immunofluorescent tool for marking endothelial cells 36 37 38 . That is the reason why, in this study, GSLI-isolectin B4 (Vector Laboratories) has been used, as described in materials and methods, to identify the number of capillaries per mm 2 in muscle area ( Figure 5F). Figure 5E).

17/ The authors should describe the details of the angiographic scoring criteria (
As requested, we give more details for the angiographic score and we added the changes highlighted in yellow in the manuscript in materials and methods, page 17, line 9-18. Mice were anesthetized (Alfaxan, 80 mg/kg body weight) and Xylazine (10 mg/kg body weight), and longitudinal laparotomy was performed to introduce a polyethylene catheter into the abdominal aorta and inject contrast medium (barium sulfate, 1 g/mL). Angiography of hindlimbs was then performed, and images (2 per animal) were acquired with the use of a high-definition digital x-ray transducer. Images were assembled to obtain a complete view of the hindlimbs. The number of pixels occupied by vessels was measured in the quantification area with the use of Primed angio software (Trophy System, Paris, France). Area of quantification was limited by placement of the iliac, the knee, the edge of the femur, and the external limit of the leg. The results were then expressed as a ratio of irradiated to nonirradiated leg.
18/ VEGF mRNA dynamics (Fig. 5B) do not match protein dynamics (Fig. 5D). Furthermore, the Vegf mRNA peaks at D90 (Fig. 5B), but the capillary density is significantly suppressed (Fig. 5F), a contradiction. The authors must explain these contradictory data. Also, the Vegf protein cannot be considered biologically meaningful if there is no significant increase or decrease (Fig. 5D).
We agree with the reviewer on the fact that there is no matching of the dynamic between mRNA and protein levels. However as previously explained for eNOS expression, the time points could be more accurate to observe the correlation. In our study, we demonstrate that VEGF mRNA expression level increase at D90, with no upregulation of VEGF protein level. This can explain the rarefaction of the vascularization. In order to not confuse the scientific message, we removed VEGF results. Fig. 5F, "cappilary"  "capillary".

19/
we modified 20/ In D3, the number of Mohi and Molo in the blood of ccr2-/-mice, but not cx3cr1-/-mice, was significantly lower than in wild-type mice (Fig. 6A), what was found and considered? The authors should also indicate whether there were changes in leukocyte infiltration into the muscle with ccr2-/-and cx3cr1-/-mice.
The role of CCR2 in the mobilization of Mo hi from the BM to the blood has been largely published 39 40 41 42 17 . In an experimental model of acute skeletal muscle injury induced by barium chloride injection, it was shown by flow cytometry that 43,44 : -at baseline, the percentage of 7/4 + Ly-6G -Mo/Mɸ was increased in bone marrow but reduced in blood in Ccr2-/-mice as compared with wild-type controls -at d3 postinjury, the percentage of 7/4 + Ly-6G -Mo/Mɸ was also increased in bone marrow and reduced in blood in Ccr2-/-mice These results highlight the role of CCR2 in the mobilization of Mo hi from the BM to the blood.
To answer to the reviewer, we analyzed the infiltration of macrophages by performing CD68 immunostaining on irradiated and non-irradiated muscle from WT, CCR2-/-and CX3CR1-/-animals, 10 days post-irradiation. We demonstrate the low number of Mɸ in CCR2-/-muscle compared to WT (p<0.05). We confirmed the role of CCR2 in the mobilization of Mo from the BM to the irradiated muscle through the blood. This result has been highlighted in the manuscript in yellow (page 10, line [10][11] and added as panel D in Figure 6 (shown below).

21/ What about BM-derived EPC infiltration, which is important for angiogenesis (DOI:
10.1172/JCI43027), into the injured muscle throughout the experimental period?
In a previous study, we evaluated the vasculogenesis process in a wound healing model in irradiated and non-irradiated condition 19 . We showed that, using chimeric GFP mice, there is a mobilization of BM-derived EPC to the site of lesion and their incorporation and differentiation into endothelial cells in the vessel (immunostaining GFP+ CD31+). However, few GFP+ CD31+ cells were identified in irradiated muscle; these results are in adequation with different studies, which have shown that vasculogenesis mechanism may be minor 45 46 . The reported relative contribution of transplanted cells to the endothelium of growing vessels varies widely, from almost no incorporation to 50% 47 48 49 .

22/ The scRNAseq experiment will address some of the questions that reviewers and readers may
have.
This study focused on physiopathology of RLI. We totally agree with the reviewer about the cellular and molecular aspects and would be necessary to investigate for a future study.

23/ Regarding the third paragraph on page 9, I wonder if a Bayesian approach is appropriate for this study. Why not actually conduct a mouse experiment and use various M1 and M2 markers to prove
it? Furthermore, the fourth paragraph is too descriptive.
We totally agree with the reviewer concerning the importance and interest of the approach based on M1 or M2 injection to prove the efficiency. However, we consider the Bayesian mathematic approach with some limitation but the easiest way to correlate Mɸ and vascularization.
However, we need to confirm the role of Mɸ on the vascularization, for example by nanoparticles injection in order to promote the M1 to M2 phenotype switch during the late phase, with the objective to stabilize the vascularization for tissue regeneration. Moreover, Mɸ experimentation typically involves ex vivo culture (e.g., from the peritoneum), or the differentiation from bone marrow progenitor cells to form bone marrow-derived macrophages. As discussed, macrophage provenance, culture conditions (L929 supernatant quality is critical), and inflammatory stimuli will naturally affect cell phenotype, function, and inflammatory status. Cell heterogeneity is a major limitation of culturing primary macrophages and it is therefore desirable to robustly control conditions in vitro in order to fully characterize the response of macrophages to specific and quantifiable stimuli 50 . In any case, the further experiments that we would provide for clinical transfer will be based on the nanoparticle administration to modulate the phenotype of the Mɸ 27,51 .
C/ Furthermore, the fourth paragraph is too descriptive.
We agree and remove the fourth paragraph There are some technical points, however, that should be addressed as well as some questions regarding some of the results: 1. There are discrepancies between figures referred to in the text and the actual figures themselves.
In particular, referring to statistical significance in the text but lacking significance markings in the graphs. Also, they sometimes refer to incorrect figure labels within the text.
a. Figure 2D is listed as 1D in text-As suggested, we modified Figure 1D to Figure 2D in the text.
More importantly, in the text (page7, line 6-9) it indicates "Mo hi, Mo lo and Neutrophils peaked at D1, D5 and D60 ", however in Figure 1 we obtained statistical significance only at D1 after IR compared to Non-irradiated (NIR) (using anova/ancova statistical test). That is the reason why no significance was marked in the graph.
As mentioned by the reviewer, we corrected in the text all the figure labels.
b. Figure 4C (T lymphocytes)-in the text they state The number of CD3 positive lymphocytes increased by 39-fold at D7, 24-fold at D14 and 18-fold at D21 however in the graph there is no D21,

only D30
As suggested by the reviewer. We modified D21 to D30.
c. Figure 6E is referred to as 6D in text As suggested, we modified Figure 6E to Figure 6D in the text.

There are acronyms such as NIR that have not been defined before their use and the manuscript
As suggested, we define in the manuscript NIR: non-irradiated and highlighted in yellow page 4, line The irradiation field is 2*24cm, hindlimb of mice is irradiated without the foot (see illustration below): The materials and methods have been completed accordingly page 17, line13-14. Estrogen are known to have a protective effect on vascular disease 52 53 54 55 and also, anti-inflammatory 56 , antioxidant 57 , and mitochondrial protective properties 58 59 . In this study, we evaluate the neovascularization and inflammatory process in tissue regeneration, that's the reason why we avoid the bias of estrogen effect on both parameters after ionizing radiation by using male mice. The radiation-induced bystander effect refers to a unique process in which factors released by irradiated cells or tissues exert effects on other parts of the animal not exposed to radiation, causing genomic instability, stress responses and altered apoptosis or cell proliferation 60 61 62 . The bystander effect can't be transferred from one animal to another animal. Moreover, in this study we didn't mix animal cages of irradiated animals and non-irradiated control littermates. As suggested by the reviewer, we clarify below and add in the discussion highlighted in yellow (page 12, line [15][16][17][18][19][20], that ionizing radiation injury is a different model of sterile injury compared to myocardial infarction. We have an increase of M2-like macrophages at D1 in irradiated muscle. In fact, we suppose that the increase of M2 like macrophages at D1 constitute a transient adaptative immune response to the tissue aggression after radiation exposure. We have several hypotheses, and it could be related to:

Material and
-The proliferation of M2 resident cells in the tissue.
-Resistance of radio induced apoptosis of M2 cells process in the muscle.
8. Furthermore, I would like them to comment on the sustained levels of neutrophils, Mo(hi) and Mo(lo) in the blood (depicted in Figure 2C) and provide a hypothesis or details on why the fluctuations in these cells observed in the spleen and muscle are not reflected in the blood.
As suggested by the reviewer, we clarify below and add in the discussion highlighted in yellow (page 12, line [11][12][13], that inflammatory cells proliferation, migration and recruitment is a dynamic process.
Two organs, bone marrow and spleen, produce innate cells and we can see the proliferation in these.
For example, at D1 post-IR there is a proliferation of innate cells, which then go to the blood and are recruited in the muscle. However, the number of innate cells in BM, spleen is around 10 6 and in the blood 10 4 , nevertheless in muscle the number of innate cells is low (25 cells at D1) compared to the level of circulating cells suggesting that there is an accumulation in the blood.

Moreover, it would have been great to see how transfer of M2-like MFs can help the healing
process after such a high radiation dose. In case the data could not be provided, the author should discuss expected outcomes after such a transfer.
we agree with the reviewer on the importance of the M2 like Mɸ injection to confirm the hypotheses and the role of Mɸ in vascular response for tissue regeneration. However, Macrophage experimentation typically involves ex vivo culture (e.g., from the peritoneum), or the differentiation from bone marrow progenitor cells to form bone marrow-derived macrophages. As discussed, macrophage provenance, culture conditions (L929 supernatant quality is critical), and inflammatory stimuli will naturally affect cell phenotype, function, and inflammatory status. Cell heterogeneity is a major limitation of culturing primary macrophages and it is therefore desirable to robustly control conditions in vitro in order to fully characterize the response of macrophages to specific and quantifiable stimuli 50 . In any case, the further experiments that we would provide for clinical transfer will be based on the nanoparticle administration to modulate the phenotype of the Mɸ 27,51 .